Strategies for Improving Soluble

Protein Production in E. coli

Key Learning Objectives

• Overview of recombinant protein expression in E. coli

• Challenges in protein expression

• Solutions

• Clone quickly

• Reduce background expression

• Fine-tune expression levels

• Overcome solubility challenges

• Express difficult membrane proteins

Why Do We Express Recombinant Proteins?For Research and Commercial Applications

To answer basic biological questions:

• In vivo: Cellular functions and mechanisms including protein-protein interactions, temporal growth studies, signal transduction, reporter assays

• In vitro: Study protein structure, function, and activity

To purify proteins for:

• Research products and services

• Industrial enzymes

• Drug discovery

• Biotherapeutics

Applications for Recombinant Proteins

Human kinases (>500)

Biotherapeutics

Industrial enzymes for consumer products

Research tools

Drug discovery

Crystal structure determination

Enzyme Classification

doi:10.1371/journal.pone.0151001.g004

E. coli is Cheapest, Quickest, Easiest SystemThe Protein Expression Host of Choice based on Pubmed

Front. Microbiol., 05 March 2014 | http://dx.doi.org/10.3389/fmicb.2014.00085

E. coli Advantages:• Well-understood genetics, easily manipulated • Easy introduction of recombinant DNA into cells• Fast, high density cell growth• Inexpensive media• Easy to scale for fermentation

Proportion of Recombinant Genes Expressed in Different Organisms

AnalysisPurification

General Protein Expression and Purification WorkflowCloning Through in vitro Analysis

Cloning Expression

Key Challenges at Each Step

• Time-consuming process

• Multiple steps where process can “go wrong”

• Many methods are not amenable to automation

• Poor clone survival with toxic targets

• Low expression levels

• Low solubility (inclusion bodies)

• Protein degradation or truncation

• Poor cell viability with toxic targets

• Need to transfer vector from cloning to expression strain

• Non-optimized purification strategies

• Occluded purification tags

• Contaminating proteins

• Ineffective cell lysis method

• Poor salt and detergent choices

• “False solubility”

• Inactive protein

• Insufficient protein yield

• Insufficient protein purity

• Endotoxin contamination

• Incorrect post-translational modifications

• Choose an expression vector

• Insert the target gene

• Transfer the cloned DNA into the host strain

• Express the protein

• Evaluate protein yield, solubility and/or activity

Protein Expression in E. coliWorkflow from Gene to Protein

Clone Target Gene

Promoter

Tag

Replication Origin

Selection Marker

Promoter

Tag

Replication Origin

Selection Marker

Target Gene

Transform E. coli

E. coli

Express Protein

Target Gene

Evaluate

Transform E. coli

Choose an Expression VectorVector Determines Downstream Strategy

Promoter

Tag

Replication Origin

Selection Marker

E. coli

Express Protein

Target Gene

Evaluate

Choice of promoter:• Inducible or constitutive?• Requires engineered expression strain?

Selection marker:• Ampicillin selection prone to satellite colonies• Carbenicillin or kanamycin less prone to satellites

Replication origin:• High or low-copy• Inducible copy number

Fusion tags:• Purification or detection• Enhance expression/solubility• Reporter

Target Gene

Tag

Replication Origin

Selection Marker

Promoter

Clone Target Gene

Selection Marker

Replication Origin Promoter Tag

Insert the Target GeneMany Cloning Technologies are Available

Other Methods:• Restriction enzyme-based (“cut and paste”)• LIC (Ligation Independent Cloning)• In-Fusion®• TOPO®• Recombinational (Gateway®)• Gibson Cloning (SGI-DNA, NEBuilder®)

PCR-amplicon Cloning Workflow

Estimated preparation time: ~10 hours

Promoter

Tag

Replication Origin

Selection Marker

Transform E. coli

E. coli

Express Protein

Target Gene

Evaluate

Clone Target Gene

Promoter

Tag

Replication Origin

Selection Marker

Target Gene

Transfer the Clone into E. coliDifferent Methods Address Different Needs

Chemically Competent Cells:• Easy to work with, less expensive

• No specialized equipment required

• Accept larger DNA volumes

• Lower transformation efficiency than Electrocompetent cells

• Used for routine cloning and plasmid propagation

Electrocompetent Cells:• Shorter protocol, amenable to

automation

• Highest transformation efficiency

• Electroporator instrument required

• Low DNA volumes with no salt required

• Used for library construction and propagation

Promoter

Tag

Replication Origin

Selection Marker

Target Gene

Tag

Replication Origin

Selection Marker

Promoter

Clone Target Gene

Transform E. coli

E. coli

Express Protein

Target Gene

Evaluate

Induce Expression and Produce ProteinSuccessful Results Depend on this Strategy

Key Variables that Determine Success: The DNA sequence

• Codon optimized for E. coli

• Optimized for secondary structure

• Target truncation

Activity of promoter Cloning & host strain capabilities

• Toxic genes, repetitive structures

• “Leaky” expression

Growth & induction conditions• Cell density at time of induction

• Length and temperature of induction

• Concentration of inducing agent

Promoter

Tag

Replication Origin

Selection Marker

Target Gene

Tag

Replication Origin

Selection Marker

Promoter

Clone Target Gene

Transform E. coli

E. coli

Express Protein

Target Gene

Evaluate

Protein Expression at the Molecular LevelMany Potential Challenges Exist

Insoluble Protein

Degraded Protein

Toxicity

Improper folding

Transmission electron microscopy

Coupled transcription/translation

Low tRNA availability

mRNA secondary structure

Translation rate too slow or too fast

Expression level too high

Origins of error:

What Can Go Wrong and Why?Issue Possible Explanations Potential Solutions

No or lowexpression

Protein may be toxic to cell before or after induction

Codon bias

• Suppress basal induction of protein- use tight promoters, defined media• Use special engineered hosts• Use tunable promoters• Reduce copy number• Direct protein to periplasm

• Optimize cDNA sequence• Use strains that are capable of supplying limited tRNAs• Increase cell density (biomass)• Use n-terminal fusion tags to overcome translational stalling

Formation of inclusion bodies

Improper folding, low solubility

Incorrect disulfide formation

Missing post-translational modification

• Fuse protein to solubility partners• Co-express molecular chaperones or use folding additives and cofactors to

medium• Remove inducer and add fresh medium• Reduce production by modifying RBS, reducing temperature, tuning inducer

concentration

• Direct protein to periplasm• Use special host with oxidative environment

• Use an alternate host

Inactive protein

Incomplete folding

Mutations in cDNA

Lacking post-translational modifications or cofactors

• Reduce growth temperature, promote disulfide bond formation

• Confirm plasmid sequence• Use recA- strains to maintain stability in cell• Use fresh transformants

• Coexpress transferases, add cofactors• Switch to yeast, insect or mammalian cell systems

Clone with quick, simple HTP-compatible method Expresso® Protein Expression Systems

Express high levels of protein with strong E. coli promoters Expresso® Rhamnose and T7 Cloning and Expression Systems

Express toxic proteins by tightly controlling expression Expresso Rhamnose Cloning and Expression System

Improve protein solubility, express toxic proteins Expresso SUMO Systems Expresso Solubility and Expression Screening System

Express membrane proteins from T7 promoter OverExpress™ C41 (DE3) and C43 (DE3) cell lines

Solutions to Common Challenges in Cloning and Expression

What is the native source and type of the protein(s) you are trying to express?

Choose all that apply.

Question #3Poll Question

AnalysisPurification

General Protein Expression and Purification WorkflowCloning Through Analysis

Cloning Expression

Key Challenges at Each Step

• Time-consuming process

• Multiple steps where process can “go wrong”

• Many methods are not amenable to automation

• Poor clone survival with toxic targets

• Low expression levels

• Low solubility (inclusion bodies)

• Protein degradation or truncation

• Poor cell viability with toxic targets

• Need to transfer vector from cloning to expression strain

• Non-optimized purification strategies

• Occluded purification tags

• Contaminating proteins

• Ineffective cell lysis method

• Poor salt and detergent choices

• “False solubility”

• Inactive protein

• Insufficient protein yield

• Insufficient protein purity

• Endotoxin contamination

• Incorrect post-translational modifications

• Simple method to clone your PCR product into a Lucigen expression vector

• Uses simple homologous recombination to fuse insert and vector

– Reaction happens inside E. coli cells, during transformation

• Speeds workflows

– Removes PCR product clean-up and ligation steps

– Reduces pipetting steps: compatible with automated screening platforms

• Directional cloning

• No cloning scars

• Uses pre-processed,

linearized vector

• Highly efficient

Simple Cloning and Expression SolutionsSpeed, Ease & High Efficiency with Expresso® Cloning

Colony PCR Shows >90% Correct Clones

Expresso® Cloning in Three Simple StepsInstant Cloning by in vivo Homologous Recombination

1. Amplify target by PCR

• Primers include ~18bp overlap with Expresso vector sequence

2. Mix PCR product and Expresso vector with competent cells

3. Transform and plate cells normally

Expresso® Workflow ComparisonFastest and Easiest PCR-based Cloning System Available

• No vector preparation

• No restriction enzymes or ligase needed

• No DNA purification steps

• Fewer handling and pipetting steps

• Compatible with automation

Expresso® Cloning Primer DesignDesign PCR Primers with Overlapping Sequences

5’ – CAT CAT CAC CAC CAT CAC – 18 – 24 nucleotides gene specific sequence

5’ – GTG GCG GCC GCT CTA TTA – 18 – 24 nucleotides reverse compliment gene specific sequence

Forward primer:

Reverse primer:

Expresso Cloning and Protein Expression Systems include:

Expresso® Systems are Complete Kits

• Linearized, dephosphorylated expression vector

• Competent cells for cloning and expression

• Control insert for cloning, expression and protease cleavage

• Primers for sequencing

• Sugar solutions for induction

• Protease enzymes for fusion tag cleavage

Which challenges are you facing in your protein expression projects?

Choose all that apply.

Poll Question

Cloning AnalysisPurification

General Protein Expression and Purification WorkflowCloning Through Analysis

Expression

Key Challenges at Each Step

• Time-consuming process

• Multiple steps where process can “go wrong”

• Many methods are not amenable to automation

• Poor clone survival with toxic targets

• Low expression levels

• Low solubility (inclusion bodies)

• Protein degradation or truncation

• Poor cell viability with toxic targets

• Need to transfer vector from cloning to expression strain

• Non-optimized purification strategies

• Occluded purification tags

• Contaminating proteins

• Ineffective cell lysis method

• Poor salt and detergent choices

• “False solubility”

• Inactive protein

• Insufficient protein yield

• Insufficient protein purity

• Endotoxin contamination

• Incorrect post-translational modifications

Codon BiasInclusion

Body Formation

Toxicity

Common Protein Expression Challenges and SolutionsKey Roadblocks to Soluble, Active protein

http://biosocialmethods.isr.umich.edu/epigenetics-tutorial/epigenetics-tutorial-gene-expression-from-dna-to-protein/

Degradation

How Can the Expresso® T7 Cloning and Expression System Help?

Challenge Lucigen Solutions

Toxicity Control basal expression with HI-Control™ cells.

Low Solubility Enhance solubility with optional SUMO tag.

Low ExpressionProduce high levels of protein from inducible T7 promoter.

Time-Consuming Cloning

Save time and increase throughput with Expresso cloning.

High-level, Inducible Expression with Low BackgroundOptimized for Tight Induction Control

Achieve High Expression Levels

• IPTG-inducible T7 promoter

Choose Optimized Vectors

• N-or C-terminal 6xHis tag

• Small: ~2.2kb

• Kanamycin resistance

• Transcriptional terminators

• Optional SUMO tag

Reduce Background Expression

• HI-Control™ cells reduce “leaky” expression during cloning and expression

• Facilitates production of toxic proteins

Transcriptional terminators stabilize clones by preventing

toxicity due to transcription into and out of cloned fragmentsT

Problem:Background expression of T7 RNA Polymerase= Uninduced target gene expression

Decreased colony count and cell viability during cloning and expression

Reduce Problematic “Leaky” ExpressionClone and Express Challenging Targets with HI-Control™

Lac Repressor

T7 Promoter Target GeneLac Operator

T7 RNA PolymeraseLac OperatorLacUV5 Promoter

Target GeneLac Operator

T7 RNA Polymerase

Induced Target Gene Expression

+ IPTG

Solution:HI-Control BL21(DE3)

HI-Control 10G (cloning strain)

Increased expression of LacIq repressor= Improved control of gene expression

Less toxicity and more soluble protein

HI-Control Cells Express LacIq Repressor

Ideal System for Routine or Toxic Proteins High-level Expression Equivalent to the pET System

Induce maximal protein expression from strong T7 promoter

Control “leaky” expression with strains expressing high levels of lac repressor (LacIq):

HI-Control™ 10G

HI-Control BL21 (DE3)

Purify protein with 6xHis tag

Clone simply with Expresso® cloning

How Can the Expresso® Rhamnose Cloning and Expression System Help?

Challenge Lucigen Solutions

ToxicityControl basal expression and fine-tune induction level of toxic proteins with tunable rhamnose-inducible promoter.

Low Solubility

Enhance solubility with optional SUMO tag.

Find optimal induction conditions with tunable rhamnose promoter to maximize soluble protein yield.

Low ExpressionFind optimal protein expression conditions using tunablerhamnose promoter.

Time-Consuming Cloning and Transfer

Save time and increase throughput with Expresso cloning.

Save time by cloning and expressing in the same strain.

Tunable Protein Expression with Virtually No BackgroundIdeal System for Toxic Proteins

Fine-Tune Expression Levels

• Rhamnose-inducible rhaPBAD promoter

• Enhanced solubility

Select Optimized Vectors

• N-or C-terminal 6xHis tag

• Small: ~2.3kb

• Kanamycin resistance

• Transcriptional terminators

• Optional SUMO tag

Express Toxic Proteins

• Eliminate “leaky” expression with glucose repression

• Express proteins in almost any E. coli cell line

Transcriptional terminators stabilize clones by preventing

toxicity due to transcription into and out of cloned fragments

Save time by using a single strain for cloning AND expression

Eliminate “leaky” expression with glucose repression

Find the best conditions for induction of toxic proteins

Easily modulate expression levels by varying rhamnose concentrations (standard induction protocol)

Combine glucose and rhamnose in the growth media for (hands-free autoinduction)

After glucose is depleted from media, cells switch to rhamnose as a carbon source

Rhamnose-induced protein expression begins

Protein Expression Levels are Responsive to Rhamnose Concentrations Between 0.001% &

0.2%

Control the Level of Protein ExpressionMaximize Soluble Protein Yields

Late autoinduction:

Start autoinduction cultures with more glucose (repressor) to delay protein expression

Build up more biomass before induction begins

Produce higher protein yields

Control the Timing of Protein ExpressionAlter Glucose Conc. for Toxic Target Autoinduction

Early Autoinduction Late Autoinduction0 6 8 10 24 0 6 8 10 24Hours

Which fusion tags have you used to express your protein in E. coli?

Choose all that apply.

Question #3Poll Question

Improve Protein Expression and SolubilityExpress Soluble, Native Proteins with Expresso® SUMO

Trusted SUMO Fusion Technology

• Small Ubiquitin-like Modifier (100 amino acid yeast protein)

• Enhance functional protein production with N-terminal SUMO tag

• Reduce likelihood of inclusion bodies (i.e. insoluble protein)

Recover Native Protein

• Cleave SUMO tag using SUMO Express Protease

• Easily remove protease with Ni2+ column purification

Select Your Promoter

• Expresso T7 SUMO Cloning and Expression System – for high-level production

• Expresso Rhamnose SUMO System – for controlled, tunable production

Recover Native Proteins QuicklySimplify Purification and Tag Cleavage

1) Express and purify tagged target protein by IMAC (Ni2+ column).

2) Add SUMO Express Protease (with 6xHis tag) and incubate.

3) Remove 6xHis-SUMO tag and SUMO Express Protease by subtractive IMAC (Ni2+ column).

4) Purified, native protein is recovered from the column flow-through.

Target ProteinSUMO

Tag6xHis

SUMO Protease

6xHis+

Target ProteinSUMO

Tag6xHis + + SUMO

Protease6xHis

+ Subtractive IMAC

Target Protein

Suen (2011) PLoS ONE 6(4):e18814.

Increase Soluble Protein YieldCleavable SUMO Tag Enhances Expression and Solubility

Gene 1 Gene 2

SUMO tag enhances solubility compared to c-terminal 6xHis tag alone

Low Recombinant Protein Solubility?Evaluation and Next Steps

M T S I Analysis by SDS-PAGE:• Grow E. coli clone(s) containing recombinant gene of interest, induce expression

• Take sample of uninduced cell culture, if applicable• Harvest cells by centrifugation• Resuspend cell pellet in sonication buffer

• Choice of protease inhibitors• Include reducing reagent (DTT)• Keep purification requirements in mind

• Lyse cells by sonication• Take sample of Total protein

• Fractionate lysate by centrifugation• Take sample of Soluble protein from supernatant• Resuspend Insoluble pellet in SDS-PAGE sample buffer

• Analyze fractions by SDS-PAGE and determine which fraction(s) contain your protein of interest.

Potential Solutions for Insoluble Proteins Impact

Purify protein as inclusion bodies. Denature and re-fold protein.

Methods are problematic. Requires high degree of skill.

Optimize protein induction parameters, export. Time-consuming, may not work.

Use optimized system with fusion partners to enhance solubility and expression levels.

Screen multiple fusion tags simultaneously with tunable promoter, easy expression protocol and fast cloning method.

How Can the Expresso® Solubility and Expression Screening System Help?

Problem Source Lucigen Solutions

Low Solubility Enhance solubility with a panel of fusion tags.

Low expression due to protein or mRNA degradation

Stabilize mRNA and protein with fusion tags.

Low expression due to codon bias

Help overcome codon bias at the 5’ end of sequence with N-terminal fusion tags. Codon bias at 5’ end often stalls translation.

ToxicityTightly control expression levels with rhamnose promoter.

Need for multiplex capabilitiesUse a high-throughput compatible cloning and screening workflow. Save time by using a single strain for cloning and expression.

Improve expression with tunable promoter

Produce native protein with SelecTEV™-cleavable tags

Purify native protein from 6xHis-SelecTEV™ Protease

Enhance Expression and Solubility with Fusion PartnersSeven Expresso® Vectors Contain a Panel of Fusion Tags

Test your amplicon with all tags in parallel:

ONLY kit with a panel of fusion tags

Day 4Day 3Day 2

Expression & Solubility Screening Workflow“Gene to Protein” in as Few as 4 Days

Designone set of PCR primers to amplify your gene.

Amplify your gene.

Clone your PCR product into all seven Expresso® fusion vectors plus the control vector.

Transform E. coli.

Evaluatetarget protein expression and solubility.

Purifyprotein (Ni2+) and remove fusion tags with SelecTEV™ Protease.

Start cultures for expression (same strain).

Day 1

Soluble, native

protein.

High Efficiency Speeds Workflows, Enables AutomationClone Into 7 Expresso® Vectors with >90% Efficiency

24 / 24 Correct Clones by Colony PCR Screen of SOL-LIN28 Target

Streamline time and effort with high efficiency cloning:

Eliminate repeat cloning attempts

Pick fewer colonies for analysis

Perform fewer minipreps, sequencing

Reduce pipetting steps, high-throughput compatible

T S I

AFVT S I

slyDT S I

tsfT S I

ControlT S I

SUMOT S I

BlaT S I

MBPT S I

GST

* * **

* * **

Quickly Identify the Best Solubility Tag for Your ProteinStreamline Screening with Parallel Processing

Results: Six of seven tags showed enhanced solubility compared to control (Lane S).Four of seven tags showed enhanced expression compared to control (Lane T), with Tsf and MBP as best performers.

T = total protein, S = soluble protein, I = insoluble proteinSMAD protein: Recruited to the TGF-b receptors and mediates TGF-b signaling

GH1 is the form of human growth hormone expressed in the pituitary gland.

Different Tags Rescue Different ProteinsBest Tag for Each Protein is Determined Empirically

T = total protein, S = soluble protein, I = insoluble protein

Results: SlyD, Tsf, SUMO, Bla, and MBP demonstrated enhanced solubility (Lane S), compared to control.SlyD, Tsf, Bla and MBP also demonstrated enhanced expression (Lane T).

• Clone the target(s) into seven fusion tag vectors, plus the control vector, which contains a 6xHis tag only.

• Compare protein expression and solubility between the control vector and the seven fusion tags.

• If you find that some tags work better than others for your proteins, we will provide those specific Expresso® fusion tag vectors as custom products (at larger scales, if desired).

• Contact custserv@Lucigen.com

Successful High-Throughput Screening with Expresso®Test Seven Tags. Re-order the Ones that Work for You.

Custom Solutions

Improve Expression of Toxic/Membrane ProteinsOverExpress® C41(DE3) & C43(DE3) Competent Cells

Mutant strains derived from BL21 (DE3) for toxic proteins (membrane proteins, cytoplasmic proteins, nucleases)

Proven success with over 350 referenced publications

Reduced expression from T7 promoters

Strains differ in tolerance for different proteins

Also available as pLysS for tighter expression control

Dumon-Seignovert, et al. , (2004). Protein Expression and Purification 37, 203-206.

Comparison of OverExpress with BL21(DE3)

Literature references: http://www.lucigen.com/OverExpress-References.html

• Design cloning primers carefully

– Check reading frame, start and stop codons, and cloning sequences

• Sequence-verify all expression clones

– Lucigen kits include sequencing primers!

– Troubleshooting: sequence-verify target in plasmid purified from expression strain

• Test more than just one colony for expression

• Sonicate cells to evaluate total protein expression (instead of lysing cell pellet in SDS-PAGE loading buffer)

– Without sonication, overexpressed protein may complex with DNA = viscous, hard-to-load sample

– The target protein may not enter the gel and won’t be visualized on the gel

• Try tunable promoter for difficult targets (Expresso® Rhamnose System)

• Optimize expression temperature and timing

– Express at 30oC or 22oC to slow down protein production

– Try different autoinduction protocols (late induction vs early induction)

• Try alternative tags, add tags to opposite termini

• Move to alternative E. coli strain

Basic Tips and Troubleshooting

Summary

If You Need To…… Expresshigh levels of protein

Express an insoluble protein

Express a toxicprotein

Improve or optimize expression conditions

Express membrane proteins from T7 promoter

Expresso® T7 Cloning and Expression System √Expresso® T7 SUMO Cloning and Expression System √ √Expresso Rhamnose Cloning and Expression System √Expresso Rhamnose SUMO Cloning and Expression System √ √Expresso Solubility and Expression Screening System √ √ √ √OverExpress™ C41(DE3) and C43(DE3) competent cells √

Resources

Poster: http://bit.ly/expresso-poster

Nature Methods: http://bit.ly/nature-methods-expresso-rhamnose

http://bit.ly/nature-methods-expresso

Application Notes:http://bit.ly/expresso-app-note

Questions? www.lucigen.com

Lucigen Tech Supporttechsupport@lucigen.com(888) 575-9695(608) 831-90118 am – 5 pm Central Time

Thank You!